Yeast Two-Hybrid Vectors

pGBKT7 is a yeast two-hybrid bait expression vector that is designed to express a fusion protein of the GAL4 DNA-binding domain (DNA-BD; amino acids 1–147) and a bait protein. The fusion protein is expressed at high levels from the ADH1 promoter. The fusion protein also contains a c-Myc epitope tag. To facilitate in vitro transcription/translation, pGBKT7 includes the T7 promoter between the GAL4 DNA-BD and the epitope tag.

pGADT7 Yeast Two-Hybrid Prey Vector

pGADT7 AD is a yeast two-hybrid expression vector that is designed to express a fusion protein between the GAL4 activation domain (AD; amino acids 768–881) and a protein of interest. The fusion protein is expressed at high levels from the full-length ADH1 promoter. The fusion protein also contains a hemagglutinin (HA) epitope tag. To facilitate in vitro transcription/translation, pGADT7 AD includes the T7 promoter between the GAL4 AD and the epitope tag.

pBridge Three-Hybrid Bait Vector

The pBridge Vector allows you to investigate tertiary protein complexes with any GAL4-based two-hybrid system. pBridge may be used instead of pGBKT7 in the Matchmaker Gold System. With its two distinct multiple cloning sites, pBridge allows expression of a DNA-BD fusion protein and another distinct protein that acts as the 'bridge' between the bait and prey (1, 2).

A three-hybrid system allows you to investigate more complex protein interactions than is possible with standard two-hybrid analysis. The third protein in this system can participate in the interaction in several ways:

Acting as a bridge, by interacting with two proteins that do not directly interact with each other

Stabilizing a weak interaction between the two proteins

Modifying or inhibiting one or both of the proteins (3)

Inhibiting the two-hybrid interaction

Expression of the additional protein is controlled by a conditional methionine promoter—it is expressed in the absence of methionine. This allows expression to be switched on or off by culturing in media with or without methionine. The effect of the third protein is indicated by expression of a reporter or nutritional marker. We offer yeast media lacking methionine (Cat. Nos. 630430 & 630429) that are ideal for use with this vector.

Features

pGBKT7 and pGADT7 are components of the Matchmaker Gold Yeast Two-Hybrid System

pBridge expresses a third protein for modified two-hybrid studies

Applications

Yeast Two-Hybrid Screening

Yeast Three-Hybrid Screening

References

Tirode, F. et al. (1997) J. Biol. Chem.272:22995–22999.

Brachmann, R. & Boeke, J. (1997) Curr. Opin. Biotechnol.8:561–568.

Osborne, M. et al. (1995) Biotechnology13:1474–1478.

Additional Information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.